The present invention relates generally to protozoan antigens and genes encoding the same. More particularly, the present invention pertains to the cloning, expression and characterization of polypeptides from Cryptosporidium parvum (C. parum) and antibodies directed to these polypeptides. The invention also pertains to use of the antigenic polypeptides and antibodies in therapeutic and diagnostic compositions.
Cryptosporidium parvum (C. parum) is a coccidian protozoan that infects a wide variety of vertebrates, including humans. C. parvum can be acquired directly from animal-to-human contact, human-to-human contact or indirectly in fomites, water and sometimes food. Current et al. (1983) N. Engl. J. Med. 308:1252-1257; Centers of Disease Control (1984) xe2x80x9cCryptosporidiosis among children attending day-care centersxe2x80x94Georgia, Pennsylvania, Michigan, California, New Mexicoxe2x80x9d 33:599-601; Wolfson et al. (1995) N. Engl. J. Med. 312:1278-1281. Acquisition of infection occurs by ingestion of oocysts which excyst in the upper small bowel releasing four infective sporozoites. The sporozoites penetrate the lining enterocyte and undergo either sexual or asexual reproduction, gametogony and merogony, respectively. The products of either form of reproduction are capable of sustaining infection in man. Navin et al. (1984) Rev. Infect. Dis. 6:313-327.
The reservoir of C. parvum is in wild and domestic animals, particularly cattle (Tzipori (1983) Microbiol. Rev. 47:84-96) and this pathogen causes significant economic losses to the farm industry annually. Clinical manifestations of C. parvum infection may include watery diarrhea, crampy epigrastric abdominal pain, malabsorption of nutrients and weight loss, anorexia and malaise. The disease is usually self limited in the immunocompetent host but can be life threatening in the immunodeficient host, particularly in human patients with advanced Human Immunodeficiency Virus (HIV) infection. Current et al. (1983), supra; Wolfson et al. (1985) N. Engl. J. Med. 312:1278-1281; Soave (1988) Infect. Dis. Clin. N. Amer. 2:485.
The absence of an adequate in vitro culture system has severely limited the investigation of C. parvum. Furthermore, use of animal models, particularly mice, is of limited value because adult mice are not normally susceptible to infection with C. parvum. 
Several groups have reported the cloning and characterization of C. parvum antigens and genes using a variety of techniques. See, e.g., Jenkins et al. (1993) Infect. Immun. 61:2377-2382; Jenkins et al. (1995) Mol. Biochem. Parasitol. 71:149-152; Peterson et al. (1992) Infect. Immun. 60:2343-2348; Ranucci et al. (1993) Infect. Immun. 61:2347-2356.
After natural infection, a wide variety of cryptosporidial proteins are recognized by human and animal immune sera. Ortega-Mora et al. (1992) Infect. Immun. 60:3442-3445; Campbell et al. (1983) J. Clin. Microbiol. 18:165-169; Whitmire et al. (1991) Infect. Immun. 59:990-995; Nina et al. (1992) Infect. Immun. 60:1509-1513; Peeters et al. (1992) Infect. Immun. 60:2309-2316. The components that constitute protective immunity are unknown although, like most obligate intracellular coccidian protozoa, both the cellular and humoral arms of the immune response are likely to play important roles in the genesis of protective immunity. Lillehoj et al. (1994) Parasit. Today 10:10-16. Use of recombinant C. parvum proteins in vaccine compositions has also been described. See, e.g., U.S. Pat. No. 5,591,434. However, no consistently effective therapy of vaccination exists, perhaps because the antigens used in the vaccines were identified from non-human sources.
There have also been several attempts to identify antibodies that neutralize C. parvum infection. In human studies, orally administered hyperimmune bovine colostrum has been found to alter the natural history of C. parvum by decreasing the excretion of oocysts, reducing the level of diarrhea, and in a smaller number of cases, clearing the parasite from stool. Tzipori et al. (1986) Br. Med. J. 293:1276-1277; Nord et al. (1989) xe2x80x9cTreatment of AIDS associated cryptosporidiosis with hyperimmune colostrum from cows vaccinated with Cryptosporidiumxe2x80x9d, Fifth International Conference on AIDS, Montreal, Quebec, May 1989; Ungar et al. (1990) Gastroenterology 98:486-489. Several antigens that are recognized by sera after natural infection have been characterized and have been shown to be the targets of neutralizing antibody in the murine system. Arrowood et al. (1989) Infect. Immun. 57:2283-2288; Bjorneby et al. (1991) Infect. Immun. 59:1172-1176; Doyle et al. (1993) Infect. Immun. 61:4079-4084; Riggs et al. (1989) J. Immunol. 143:1340-1345; Peterson et al. (1992) Infect. Immun. 60:2343-2348. However, animal studies in the murine model of infection indicate only a partially protective role when these antibodies are orally administered.
Thus, there remains a need for the identification of antibodies useful in diagnostic and therapeutic compositions and for the identification of antigens from C. parvum that react with human sera and for diagnostic and therapeutic compositions and methods using these antigens.
The present invention is based on the discovery of genes encoding C. parvum antigenic polypeptides, the characterization of these polypeptides and antibodies that recognize epitopes of these polypeptides. The proteins encoded by the genes have been recombinantly produced and these polypeptides, immunogenic fragments and analogs thereof, and/or chimeric proteins including the same, can be used, either alone or in combination with other C. parvum antigens, in novel subunit vaccines to provide protection from cryptosporidial infection in mammalian subjects. Antibodies generated against these proteins, and/or fragments thereof, either alone or in combination with other therapeutic agents, can be used in novel therapeutic agents for mammalian subjects. Furthermore, the antigens and antibodies can be used as diagnostics.
Accordingly, in one embodiment, the subject invention is directed to an isolated nucleic acid molecule comprising a coding sequence for an immunogenic C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1 and (b) a C. parvum antigenic polypeptide AG2, or a fragment of the nucleic acid molecule comprising at least 15 nucleotides.
In additional embodiments, the invention is directed to recombinant vectors including the nucleic acid molecules, host cells transformed with these vectors, and methods of recombinantly producing C. parvum antigenic polypeptides.
In still further embodiments, the subject invention is directed to vaccine compositions comprising a pharmaceutically acceptable vehicle and an immunogenic C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1, (b) a C. parvum antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, as well as methods of preparing the vaccine compositions.
In other embodiments, the invention is directed to therapeutic compositions comprising a pharmaceutically acceptable vehicle and an antibody (e.g., monoclonal antibody 1101 or 222) that recognize an immunogenic C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1, (b) a C. parvum antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, as well as methods of preparing the therapeutic compositions.
In yet other embodiments, the present invention is directed to methods of treating or preventing C. parvum infections in a mammalian subject. The method comprises administering to the subject a therapeutically effective amount of the above vaccine or therapeutic compositions.
In additional embodiments, the invention is directed to methods of detecting C. parvum antibodies in a biological sample comprising:
(a) providing a biological sample;
(b) reacting the biological sample with a C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1, (b) a C. parvum antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, under conditions which allow C. parvum antibodies, when present in the biological sample, to bind to the C. parvum antigenic polypeptide to form an antibody/antigen complex; and
(c) detecting the presence or absence of the complex,
thereby detecting the presence or absence of C. parvum antibodies in the sample.
In additional embodiments, the invention is directed to methods of detecting C. parvum antigens in a biological sample comprising:
(a) providing a biological sample;
(b) reacting the biological sample with an antibody that recognizes a C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1, (b) a C. parvum antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, under conditions which allow C. parvum antigens, when present in the biological sample, to bind to the C. parvum antibody to form an antibody/antigen complex; and
(c) detecting the presence or absence of the complex,
thereby detecting the presence or absence of C. parvum antigens in the sample.
In yet further embodiments, the invention is directed to an immunodiagnostic test kit for detecting C. parvum infection. The test kit comprises a C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1, (b) a C. parvum antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, and instructions for conducting the immunodiagnostic test. The invention is also directed to immunodiagnostic test kits comprising an antibody that recognizes a C. parvum antigenic polypeptide selected from the group consisting of (a) a C. parvum antigenic polypeptide AG1, (b) a C. parvum antigenic polypeptide AG2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, and instructions for conducting the immunodiagnostic test.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.